Fused ring heterocycles as potassium channel modulators

ABSTRACT

Compounds, compositions and methods are provided which are useful in the treatment of diseases through the modulation of potassium ion flux through voltage-dependent potassium channels. More particularly, the invention provides quinazolinone, compositions and methods that are useful in the treatment of central or peripheral nervous system disorders (e.g., migraine, ataxia, Parkinson&#39;s disease, bipolar disorders, trigeminal neuralgia, spasticity, mood disorders, brain tumors, psychotic disorders, myokymia, seizures, epilepsy, hearing and vision loss, Alzheimer&#39;s disease, age-related memory loss, learning deficiencies, anxiety and motor neuron diseases, maintaining bladder control or treating urinary incontinence) and as neuroprotective agents (e.g., to prevent stroke and the like) by modulating potassium channels associated with the onset or recurrence of the indicated conditions.

FIELD OF THE INVENTION

This invention relates to the use of certain fused ring heterocycles aspotassium channel modulators and to the treatment of diseases in which apotassium channel is implicated. Additionally, this invention relates tonovel compounds that are useful as potassium channel modulators.

BACKGROUND OF THE INVENTION

Ion channels are cellular proteins that regulate the flow of ions,including calcium, potassium, sodium and chloride, into and out ofcells. These channels are present in all human cells and affect suchprocesses as nerve transmission, muscle contraction and cellularsecretion. Among the ion channels, potassium channels are the mostubiquitous and diverse, being found in a variety of animal cells such asnervous, muscular, glandular, immune, reproductive, and epithelialtissue. These channels allow the flow of potassium in and/or out of thecell under certain conditions. For example, the outward flow ofpotassium ions upon opening of these channels makes the interior of thecell more negative, counteracting depolarizing voltages applied to thecell. These channels are regulated, e.g., by calcium sensitivity,voltage-gating, second messengers, extracellular ligands, andATP-sensitivity.

Potassium channels are associated with a number of physiologicalprocesses, including regulation of heartbeat, dilation of arteries,release of insulin, excitability of nerve cells, and regulation of renalelectrolyte transport. Potassium channels are made by alpha subunitsthat fall into at least 8 families, based on predicted structural andfunctional similarities (Wei et al., Neuropharmacology 35(7): 805-829(1997)). Three of these families (Kv, eag-related, and KQT) share acommon motif of six transmembrane domains and are primarily gated byvoltage. Two other families, CNG and SK/IK, also contain this motif butare gated by cyclic nucleotides and calcium, respectively. The threeother families of potassium channel alpha subunits have distinctpatterns of transmembrane domains. Slo family potassium channels, or BKchannels have seven transmembrane domains (Meera et al., Proc. Natl.Acad. Sci. U.S.A. 94(25): 14066-71 (1997)) and are gated by both voltageand calcium or pH (Schreiber et al., J. Biol. Chem. 273: 3509-16(1998)). Another family, the inward rectifier potassium channels (Kir),belongs to a structural family containing two transmembrane domains, andan eighth functionally diverse family (TP, or “two-pore”) contains twotandem repeats of this inward rectifier motif.

Potassium channels are typically formed by four alpha subunits, and canbe homomeric (made of identical alpha subunits) or heteromeric (made oftwo or more distinct types of alpha subunits). In addition, potassiumchannels made from Kv, KQT and Slo or BK subunits have often been foundto contain additional, structurally distinct auxiliary, or beta,subunits. These subunits do not form potassium channels themselves, butinstead they act as auxiliary subunits to modify the functionalproperties of channels formed by alpha subunits. For example, the Kvbeta subunits are cytoplasmic and are known to increase the surfaceexpression of Kv channels and/or modify inactivation kinetics of thechannel (Heinemann et al., J. Physiol. 493: 625-633 (1996); Shi et al.,Neuron 16(4): 843-852 (1996)). In another example, the KQT family betasubunit, minK, primarily changes activation kinetics (Sanguinetti etal., Nature 384: 80-83 (1996)).

Slo or BK potassium channels are large conductance potassium channelsfound in a wide variety of tissues, both in the central nervous systemand periphery. They play a key role in the regulation of processes suchas neuronal integration, muscular contraction and hormone secretion.They may also be involved in processes such as lymphocytedifferentiation and cell proliferation, spermatocyte differentiation andsperm motility. Three alpha subunits of the Slo family have been cloned,i.e., Slo1, Slo2, and Slo3 (Butler et al., Science 261: 221-224 (1993);Schreiber et at, J. Biol. Chem., 273: 3509-16 (1998); and Joiner et al.,Nature Neurosci. 1: 462-469 (1998)). These Slo family members have beenshown to be voltage and/or calcium gated, and/or regulated byintracellular pH.

Certain members of the Kv family of potassium channels were recentlyrenamed (see, Biervert, et al., Science 279: 403-406 (1998)). KvLQT1 wasre-named KCNQ1, and the KvLQT1-related channels (KvLR1 and KvLR2) wererenamed KCNQ2 and KCNQ3, respectively. More recently, additional membersof the KCNQ subfamily were identified. For example, KCNQ4 was identifiedas a channel expressed in sensory outer hair cells (Kubisch, et al.,Cell 96(3): 437-446 (1999)). KCNQ5 (Kananura et al., Neuroreport11(9):2063 (2000)), KCNQ 2/3 (Main et al., Mol. Pharmacol. 58: 253-62(2000), KCNQ 3/5 (Wickenden et al., Br. J. Pharma 132: 381 (2001)) andKCNQ6 have also recently been described.

KCNQ2 and KCNQ3 have been shown to be nervous system-specific potassiumchannels associated with benign familial neonatal convulsions (“BFNC”),a class of idiopathic generalized epilepsy (see, Leppert, et al., Nature337: 647-648 (1989)). These channels have been linked to M-currentchannels (see, Wang, et al., Science 282: 1890-1893 (1998)). Thediscovery and characterization of these channels and currents providesuseful insights into how these voltage dependent (Kv) potassium channelsfunction in different environments, and how they respond to variousactivation mechanisms. Such information has now led to theidentification of modulators of KCNQ2 and KCNQ3 potassium channels orthe M-current, and the use of such modulators as therapeutic agents. Themodulators are the subject of the present invention.

SUMMARY OF THE INVENTION

The present invention provides fused ring heterocycles andpharmaceutically acceptable salts thereof (“compounds of theinvention”), which are useful in the treatment of diseases through themodulation of potassium ion flux through voltage-dependent potassiumchannels.

In one aspect, the present invention provides compounds of the formula:

in which the symbol A represents a ring system such as a five- orsix-membered substituted or unsubstituted aryl, a five- and six-memberedsubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedC₄-C₈ cycloalkyl, or a substituted or unsubstituted 5-8 memberedheterocyclyl.

In an exemplary embodiment, A is substituted or unsubstituted phenyl.Exemplary substituted phenyl moieties include those that are substitutedwith one or two groups that are independently selected from halogen,nitrile, substituted or unsubstituted C₁-C₄ alkyl, SCF₃, trifluoromethyland trifluoromethoxy.

X is a moiety such as CO, CS or SO₂. The symbol W represents N or CR³,in which R³ is H, F, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 5-7 membered heterocyclyl, or substitutedor unsubstituted C₁-C₈ alkyl.

The symbol Z indicates a bond, —CH₂—, —CHF—, —CF₂—, —CH═CH— or—NR⁴(CR^(4a)R^(4b))_(s)—, wherein R⁴ is a member selected from H and asubstituted or unsubstituted C₁-C₅ alkyl group. The symbols R^(4a) andR^(4b) represent groups that are independently selected from H,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted C₃-C₈ cycloalkyl, substitutedor unsubstituted 5-7 membered heterocyclyl, or substituted orunsubstituted C₁-C₈ alkyl. The symbol s represents an integer from 1 to3; Y represents (CR⁵R⁶)_(n), in which n is an integer from 0-4. In apreferred embodiment, when n is 0 and R² is methyl, A is not anunsubstituted phenyl moiety.

R⁵ and R⁶ independently represent H, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedC₃-C₈ cycloalkyl, substituted or unsubstituted 5-7 memberedheterocyclyl, or substituted or unsubstituted C₁-C₈ alkyl.

The symbol R¹ represents a moiety that is selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 5-7membered heterocyclyl, and substituted or unsubstituted C₁-C₈ alkyl.

In an exemplary embodiment, R¹ is substituted or unsubstituted phenyl.When R¹ is substituted phenyl, it is preferably substituted by one ormore independently selected moiety, such as halogen, CF₃ or OCF₃.

R² is CF₃, substituted or unsubstituted C₁-C₈ alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted3-7-membered heterocyclyl. R² is preferably a substituted orunsubstituted C₁-C₆ saturated acyclic alkyl group, more preferably aC₁-C₄ saturated acyclic alkyl group.

In another aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and acompound of the formula provided above.

In yet another aspect, the present invention provides a method forincreasing flow through voltage dependent potassium channels in a cell,comprising contacting the cell with a compound of the formula providedabove in an amount sufficient to open the potassium channels.

In still another aspect, the present invention provides a method fortreating a central or peripheral nervous system disorder or conditionthrough the modulation of a voltage-dependent potassium channel, themethod comprising administering to a subject in need of such treatmentan effective amount of a compound of the formula provided above.

Other objects and advantages of the present invention will be apparentfrom the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays structures of representative compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

Abbreviations and Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. For example: CHO, Chinese hamster ovary;EBSS, Earl's Balanced Salt Solution; KCNQ, potassium channel Q; KCNQ2,potassium channel Q2, hSK, Ca²⁺ activated small conductance potassiumchannels; SDS, sodium dodecyl sulfate; Et₃N, triethylamine; MeOH,methanol; and DMSO, dimethylsulfoxide.

“Compound of the invention,” as used herein refers to a compoundaccording to Formulae (I)-(V) or a combination thereof, and apharmaceutically acceptable salt of a compound according to Formulae(I)-(V) or a combination thereof.

“Modulating,” as used herein, refers to the ability of a compound of theinvention to activate and/or inhibit a potassium channel, preferably, aKCNQ potassium channel.

“Opening” and “activating” are used interchangeably herein to refer tothe partial or full activation of a KCNQ channel by a compound of theinvention, which leads to an increase in ion flux either into or out ofa cell in which a KCNQ channel is found.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—; —NHS(O)₂— is also intended to represent. —S(O)₂HN—, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2m′+1), where m′ is the total number of carbon atoms in suchradical. R′, R″, R′″ and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, e.g., aryl substituted with 1-3 halogens,substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. When a compound of the invention includes more thanone R group, for example, each of the R groups is independently selectedas are each R′, R″, R′″ and R″″ groups when more than one of thesegroups is present. When R′ and R″ are attached to the same nitrogenatom, they can be combined with the nitrogen atom to form a 5-, 6-, or7-membered ring. For example, —NR′R″ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussionof substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ′N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′ or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The symbol

denotes a point of attachment of a moiety to the remainder of amolecule.

Introduction

The present invention provides compounds which, inter alia, are usefulin the treatment of diseases through the modulation of potassium ionflux through voltage-dependent potassium channels. More particularly,the invention provides compounds, compositions and methods that areuseful in the treatment of central or peripheral nervous systemdisorders (e.g., migraine, ataxia, Parkinson's disease, bipolardisorders, trigeminal neuralgia, spasticity, mood disorders, braintumors, psychotic disorders, myokymia, seizures, epilepsy, hearing andvision loss, Alzheimer's disease, age-related memory loss, learningdeficiencies, anxiety and motor neuron diseases), and as neuroprotectiveagents (e.g., to prevent stroke and the like). Compounds of theinvention have use as agents for treating convulsive states, for examplethat following grand mal, petit mal, psychomotor epilepsy or focalseizure. The compounds of the invention are also useful in treatingdisease states such as gastroesophogeal reflux disorder andgastrointestinal hypomotility disorders.

The compounds of the invention are also useful in the treatment,prevention, inhibition and amelioration of urge urinary incontinencealso known as bladder instability, neurogenic bladder, voidingdysfunction, hyperactive bladder or detrusor overactivity. The methodsof this invention also include the prevention and treatment of mixedstress and urge urinary incontinence, including that associated withsecondary conditions such as prostate hypertrophy. The methods of thisinvention are useful for inducing, assisting or maintaining desirablebladder control in a mammal experiencing or susceptible to bladderinstability or urinary incontinence. These methods include prevention,treatment or inhibition of bladder-related urinary conditions andbladder instability, including idiopathic bladder instability, nocturnalenuresis, nocturia, voiding dysfunction and urinary incontinence. Alsotreatable or preventable with the methods of this invention is bladderinstability secondary to prostate hypertrophy. The compounds describedherein are also useful in promoting the temporary delay of urinationwhenever desirable. The compounds of this invention may also be utilizedto stabilize the bladder and treat or prevent incontinence which urgeurinary incontinence, stress urinary incontinence or a combination ofurge and stress incontinence in a mammal, which may also be referred toas mixed urge and stress incontinence. These methods include assistancein preventing or treating urinary incontinence associated with secondaryconditions such as prostate hypertrophy. These methods may be utilizedto allow a recipient to control the urgency and frequency of urination.The methods of this invention include the treatment, prevention,inhibition and amelioration of urge urinary incontinence also known asbladder instability, neurogenic bladder, voiding dysfunction,hyperactive bladder, detrusor overactivity, detrusor hyper-reflexia oruninhibited bladder.

As described above, methods of this invention include treatments,prevention, inhibition or amelioration of hyperactive or unstablebladder, neurogenic bladder, sensory bladder urgency, or hyperreflexicbladder. These uses include, but are not limited to, those for bladderactivities and instabilities in which the urinary urgency is associatedwith prostatitis, prostatic hypertrophy, interstitial cystitis, urinarytract infections or vaginitis. The methods of this invention may also beused to assist in inhibition or correction of the conditions ofFrequency-Urgency Syndrome, and lazy bladder, also known as infrequentvoiding syndrome. The methods of this invention may also be used totreat, prevent, inhibit, or limit the urinary incontinence, urinaryinstability or urinary urgency associated with or resulting fromadministrations of other medications, including diuretics, vasopressinantagonists, anticholinergic agents, sedatives or hypnotic agents,narcotics, alpha-adrenergic agonists, alpha-adrenergic antagonists, orcalcium channel blockers.

Moreover, compounds of the invention are useful in the treatment ofpain, for example, neuropathic pain, inflammatory pain, cancer pain,migraine pain, and musculoskeletal pain. The compounds are also usefulto treat conditions, which may themselves be the origin of pain, forexample, inflammatory conditions, including arthritic conditions (e.g.,rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and goutyarthritis) and non-articular inflammatory conditions (e.g., herniated,ruptured and prolapsed disc syndrome, bursitis, tendonitis,tenosynovitis, fibromyalgia syndrome, and other conditions associatedwith ligamentous sprain and regional musculoskeletal strain).Particularly preferred compounds of the invention are less ulcerogenicthan other anti-inflammatory agents (e.g., ibuprofen, naproxen andaspirin). Furthermore, the compounds of the invention are useful intreating conditions and pain associated with abnormally raised skeletalmuscle tone.

The compounds of the invention are also of use in treating anxiety (e.g.anxiety disorders). Anxiety disorders are defined in the Diagnostic andStatistical Manual of Mental Disorders (Third Edition-revised 1987,published by the American Psychiatric Association, Washington, D.C.,see, pages 235 to 253), as psychiatric conditions having symptoms ofanxiety and avoidance behavior as characteristic features. Includedamongst such disorders are generalized anxiety disorder, simple phobiaand panic disorder.

Anxiety also occurs as a symptom associated with other psychiatricdisorders, for example, obsessive compulsive disorder, post-traumaticstress disorder, schizophrenia, mood disorders and major depressivedisorders, and with organic clinical conditions including, but notlimited to, Parkinson's disease, multiple sclerosis, and otherphysically incapacitating disorders.

In view of the above-noted discovery, the present invention providescompounds, compositions, and methods for increasing ion flux involtage-dependent potassium channels, particularly those channelsresponsible for the M-current. As used herein, the term “M-current,”“channels responsible for the M-current” and the like, refers to aslowly activating, non-inactivating, slowly deactivating voltage-gatedK⁺ channel. M-current is active at voltages close to the threshold foraction potential generation in a wide variety of neuronal cells, andthus, is an important regulator of neuronal excitability.

Recently, members of the voltage-dependent potassium channel family wereshown to be directly involved in diseases of the central or peripheralnervous system. The fused ring heterocycles provided herein are nowshown to act as potassium channel modulators.

Description of the Embodiments

I. Modulators of Voltage-Dependent Potassium Channels

In one aspect, the present invention provides compounds of the formula:

in which the symbol A represents a ring system such as a five- orsix-membered substituted or unsubstituted aryl, a five- and six-memberedsubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedC₄-C₈ cycloalkyl, or a substituted or unsubstituted 5-8 memberedheterocyclyl.

In an exemplary embodiment, A is substituted or unsubstituted phenyl.Exemplary substituted phenyl moieties include those that are substitutedwith one or two groups that are independently selected from halogen,nitrile, substituted or unsubstituted C₁-C₄ alkyl, SCF₃, trifluoromethyland trifluoromethoxy. In another exemplary embodiment, A is substitutedor unsubstituted 5- or 6-membered heteroaryl.

X is a moiety such as CO, CS or SO₂. The symbol W represents N or CR³,in which R³ is H, F, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 5-7 membered heterocyclyl, or substitutedor unsubstituted C₁-C₈ alkyl.

The symbol Z indicates a bond, —CH₂—, —CHF—, —CF₂—, —CH═CH— or—NR⁴(CR^(4a)R^(4b))_(s)—, wherein R⁴ is a member selected from H and asubstituted or unsubstituted C₁-C₅ alkyl group. The symbols R^(4a) andR^(4b) represent groups that are independently selected from H,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted C₃-C₈ cycloalkyl, substitutedor unsubstituted 5-7 membered heterocyclyl, or substituted orunsubstituted C₁-C₈ alkyl. The symbol s represents an integer from 1 to3; Y represents (CR⁵R⁶)_(n), in which n is an integer from 0-4. In apreferred embodiment, when n is 0 and R² is methyl, A is not anunsubstituted phenyl moiety.

R⁵ and R⁶ independently represent H, F, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 5-7membered heterocyclyl, or substituted or unsubstituted C₁-C₈ alkyl.

The symbol R¹ represents a moiety that is selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 5-7membered heterocyclyl, and substituted or unsubstituted C₁-C₈ alkyl.

In an exemplary embodiment, R¹ is substituted or unsubstituted phenyl.When R¹ is substituted phenyl, it is preferably substituted by one ormore independently selected moiety, such as substituted or unsubstitutedC₁-C₈ alkyl, substituted or unsubstituted 2 to 8 membered heteroalkyl,halogen, CN, CF₃ or OCF₃.

R² is CF₃, substituted or unsubstituted C₁-C₈ alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted3-7-membered heterocyclyl. R² is preferably a substituted orunsubstituted C₁-C₆ acyclic alkyl group, more preferably a C₁-C₄saturated acyclic alkyl group or CF₃. In some related embodiments, Y is—CF₂— or n is 0.

In an exemplary embodiment, when X is C(O); and A is unsubstitutedphenyl or 1,3-benzodioxolyl or 6-halophenyl; and W is N; and Y—R² isunsubstituted acyclic alkyl, acyclic alkyl substituted with an amide orunsubstituted phenyl, then Z—R¹ is other than acyclic alkyl substitutedwith unsubstituted phenyl, acyclic alkylene substituted withunsubstituted phenyl and acyclic alkyl substituted with 4-phenyl-1-halo.In preferred embodiments of compounds according to the above proviso,Y—R² is acyclic C₁-C₄ linear alkyl or C₁-C₄ branched alkyl, e.g.,methyl, ethyl, and isopropyl. In still further preferred embodimentsaccording to the above proviso, Z—R¹ is other than unsubstituted benzyl,unsubstituted phenethyl. In another preferred embodiment, Z—R¹ is otherthan —CH═CH-phenyl.

In another exemplary embodiment, A is selected from substituted orunsubstituted pyrazolyl, substituted or unsubstituted imidazolyl,substituted or unsubstituted thiazolyl, substituted or unsubstitutedoxazolyl, substituted or unsubstituted triazolyl, substituted orunsubstituted isothiazolyl, substituted or unsubstituted isoxazolyl, andsubstituted or unsubstituted 1,2,3,-oxadiazolyl. A may also be selectedfrom substituted or unsubstituted pyrazolyl or substituted orunsubstituted imidazolyl.

In a related embodiment, A is substituted with R⁸ and R⁹, wherein R⁸ andR⁹ are independently selected from: H, halo, CF₃, CF₃O, NO₂, CN,S(O)_(m)R¹⁰, COOR¹¹, CONR¹²R¹³, SO₂NR¹²R¹³, S(O)_(m)CF₃, CH₂CF₃,substituted or unsubstituted C₁-C₆ alkyl, and substituted orunsubstituted C₃-C₇ cycloalkyl. R¹⁰ and R¹¹ are independently selectedfrom substituted or unsubstituted C₁-C₅ alkyl, and substituted orunsubstituted C₃-C₇ cycloalkyl. The symbol m represents an integer from0 to 2. R¹² and R¹³ are independently selected from H, substituted orunsubstituted C₁-C₅ alkyl, and substituted or unsubstituted C₃-C₇cycloalkyl. R¹² and R¹³ are optionally joined together with the nitrogenatom to which they are attached to form a 5- to 7-membered ring.

In some embodiments, A has the formula:

In Formula (II), E is CR⁹ and G is N, or E is N and G is CR⁹. R⁸ and R⁹may be independently selected from CF₃, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted C₃-C₇ cycloalkyl, OCF₃ andCH₂CF₃.

In other embodiments, A has the formula:

In Formula (III), E is selected from CR⁹ and N, and G is selected from Oand S. R⁸ and R⁹ are independently selected from CF₃, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₇cycloalkyl, OCF₃, and CH₂CF₃.

In exemplary embodiments related to compounds with A having Formula (II)or (III), X is a member selected from CO and SO₂; Z is CH₂; W is N; andR¹ is substituted or unsubstituted phenyl. In other related embodiments,R² is a member selected from substituted or unsubstituted C₁-C₄ alkyl,substituted or unsubstituted C₃-C₆ cycloalkyl and substituted orunsubstituted C₃-C₆ heterocyclyl. R⁸ and R⁹ may be independentlyselected from H, halo, CF₃, OCF₃, substituted or unsubstituted C₁-C₅alkyl, SCF₃, CH₂CF₃ and CN. Y may be —CF₂— or n is 0.

In another exemplary embodiment, the invention provides a compoundhaving the formula:

in which the symbols R⁸ and R⁹ independently represent H, halo, CF₃,CF₃O, NO₂, CN, S(O)_(m)R¹⁰, COOR¹¹, CONR¹²R¹³, SO₂NR¹²R¹³, S(O)_(m)CF₃,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted C₃-C₇ cycloalkyl. R¹⁰ and R¹¹ independently representsubstituted or unsubstituted C₁-C₅ alkyl, or substituted orunsubstituted C₃-C₇ cycloalkyl. The symbol m represents an integer from0 to 2. R¹² and R¹³ are independently H, substituted or unsubstitutedC₁-C₅ alkyl, or substituted or unsubstituted C₃-C₇ cycloalkyl. R¹² andR¹³, together with the nitrogen atom to which they are attached,optionally form a 5- to 7-membered ring. The identities of the remainingvariable groups are substantially identical to their counterpartsdiscussed hereinabove.

In an exemplary embodiment, R⁸ and R⁹ are independently selected from H,halo, CF₃, OCF₃, substituted or unsubstituted C₁-C₅ alkyl, SCF₃, CH₂CF₃and CN. In another exemplary embodiment, Y is —CF₂— or n is 0. R² may bea member selected from substituted or unsubstituted C₁-C₄ alkyl,substituted or unsubstituted C₁-C₆ cycloalkyl and substituted orunsubstituted C₁-C₆ heterocyclyl.

Exemplary compounds of the invention in which X is C(O); R⁸ and R⁹ areboth H or R⁸ is 6-halophenyl and R⁹ is H or R⁸ and R⁹ taken togetherwith the carbon atoms to which they are joined form a dioxolyl ring; Wis N; and Y—R² is unsubstituted acyclic alkyl, acyclic alkyl substitutedwith an amide or unsubstituted phenyl, have a Z—R¹ group that is otherthan acyclic alkyl substituted with unsubstituted phenyl, acyclicalkylene substituted with unsubstituted phenyl and acyclic alkylsubstituted with 4-phenyl-1-halo.

Another exemplary embodiment of the invention provides compounds havingthe formula:

in which the identity of each variable moiety is substantially identicalto its counterpart discussed above in reference to Formula (IV).

In certain preferred compounds according to Formulae (IV) and (V), R² isa member selected from substituted or unsubstituted C₁-C₄ alkyl,substituted or unsubstituted C₁-C₆ cycloalkyl and substituted orunsubstituted C₁-C₆ heterocyclyl; and R⁸ and R⁹ are optionally membersindependently selected from H, halo, CF₃, OCF₃, substituted orunsubstituted C₁-C₅ alkyl, SCF₃, CH₂CF₃ and CN. In another exemplaryembodiment, Y is —CF₂— or n is 0. In related embodiments, X is CO; R⁸ isH; R⁹ is CF₃; n is 0; R² is C₁-C₄ alkyl; and R¹ is phenyl substitutedwith a halo.

Exemplary compounds of the invention have Z—R¹ groups that are otherthan acyclic alkyl substituted with unsubstituted phenyl, acyclicalkylene substituted with unsubstituted phenyl and acyclic alkylsubstituted with 4-phenyl-1-halo when X is C(O); R⁸ and R⁹ are both H; Wis N; and Y—R² is unsubstituted acyclic alkyl, acyclic alkyl substitutedwith an amide or unsubstituted phenyl.

Also within the scope of the present invention are compounds of theinvention that are poly- or multi-valent species, including, forexample, species such as dimers, trimers, tetramers and higher homologsof the compounds of the invention or reactive analogues thereof. Thepoly- and multi-valent species can be assembled from a single species ormore than one species of the invention. For example, a dimeric constructcan be “homo-dimeric” or “heterodimeric.” Moreover, poly- andmulti-valent constructs in which a compound of the invention, or areactive analogue thereof, is attached to an oligomeric or polymericframework (e.g., polylysine, dextran, hydroxyethyl starch and the like)are within the scope of the present invention. The framework ispreferably polyfunctional (i.e. having an array of reactive sites forattaching compounds of the invention). Moreover, the framework can bederivatized with a single species of the invention or more than onespecies of the invention.

Moreover, the present invention includes compounds within the motif setforth in Formula I, which are functionalized to afford compounds havinga water-solubility that is enhanced relative to analogous compounds thatare not similarly functionalized. Methods of enhancing thewater-solubility of organic compounds are known in the art. Such methodsinclude, but are not limited to, functionalizing an organic nucleus witha permanently charged moiety, e.g., quaternary ammonium, or a group thatis charged at a physiologically relevant pH, e.g. carboxylic acid,amine. Other methods include, appending to the organic nucleus hydroxyl-or amine-containing groups, e.g. alcohols, polyols, polyethers, and thelike. Representative examples include, but are not limited to,polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art. See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991.

Preparation of Potassium Channel Modulators

The method by which the compounds of the invention are prepared is notcritical; any synthetic pathway that leads to the desired compounds isof use to prepare fused ring heterocycles that are within the scope ofthe invention set forth herein.

In an exemplary reaction pathway, fused ring heterocycles of theinvention are prepared by cyclizing an anthranilic acid. Substitutedanthranilic acid precursors were prepared and cyclized and the cyclizedproducts optionally elaborated following the method outlined in Scheme1.

Substituted aniline a is chain-extended to form the intermediateα-oximinoanilides b using chloral hydrate and hydroxylamine. Treatmentof these intermediates with strong acid at elevated temperaturesfacilitates the desired ring closure to generate the substituted isatinsc. Selective ring opening of the isatins using sodium hydroxide andhydrogen peroxide provides the desired anthranilic acids d. According tothe present scheme, a two-step one-pot procedure is used to convert thesubstituted anthranilic acids into the versatile substituted3-amino-3H-quinazolin-4-ones e. The final products f and g were obtainedby either acylating the free amino group to generate the appropriateamides or by reacting the free amine with an isocyanate to yield thecorresponding urea.

The 3-amino-substituted fused ring heterocycles of the invention arereadily formed by methods such as that set forth in Scheme 2 in which dis cyclized to the corresponding quinazolinone e.

The amine moiety can be elaborated by methods such as that set forth inScheme 3, showing a general preparative route for converting a3-aminoquinazolinone into the corresponding urea quinazolinone g.

The amine moiety of the 3-aminoquinazolinone e is also readily convertedto the corresponding amide f according to Scheme 4.

Alternatively, the amides of the invention are prepared according to theroute set forth in Scheme 5.

As shown in Scheme 6, another useful synthetic route to quinazolinoneamides begins with an anthranilic acid d, which is converted to isatoicanhydride. The corresponding acylhydrazide k is produced by opening thecyclic anhydride with the desired hydrazide. Quinazolinone amide f isformed by forming a ring between an amine of the hydrazide moiety andthe aniline nitrogen.

In an alternate route, also according to Scheme 6, the carboxylic acidof nitrobenzoic acid i is activated and the activated agent used toacylate a hydrazide, forming nitrobenzyl hydrazide j, the nitro group ofwhich is reduced, forming the corresponding acylhydrazide k.

The 1,1-dioxo-1H-1λ⁶-benzo[1,2,4]thiadiazin-2-yl compounds of theinvention can be prepared by the method of Scheme 7, beginning with anitrosulfonyl chloride l. The chloride is displaced by a hydrazide,forming m. The nitro group is reduced, providing aniline n, the amine ofwhich is subsequently displaced, forming o.

Methods for preparing dimers, trimers and higher homologs of smallorganic molecules, such as those of the present invention, as well asmethods of functionalizing a polyfunctional framework molecule are wellknown to those of skill in the art. For example, an aromatic amine ofthe invention is converted to the corresponding isothiocyanate by theaction of thiophosgene. The resulting isothiocyanate is coupled to anamine of the invention, thereby forming either a homo- or heterodimericspecies. Alternatively, the isothiocyanate is coupled with anamine-containing backbone, such as polylysine, thereby forming aconjugate between a polyvalent framework and a compound of theinvention. If it is desired to prepare a hetereofuntionalized polyvalentspecies, the polylysine is underlabeled with the first isothiocyanateand subsequently labeled with one or more different isothiocyanates.Alternatively, a mixture of isothiocyanates is added to the backbone.Purification proceeds by, for example, size exclusion chromatography,dialysis, nanofiltration and the like.

Exemplary methods for producing 5-membered fused ring heterocycles arepresented in Example 6 below. One of skill in the art will immediatelyrecognize that compounds having a wide variety of 5-memberedheterocycles may be synthesized by elaboration of the disclosedsynthesis methods.

In Schemes 1-7 above and 8-15 below, the symbol X represents at least onmoiety equivalent to R⁸ and/or R⁹ as discussed above; the symbol X″represents at least one moiety independently selected from substitutedor unsubstituted C₁-C₈ alkyl, substituted or unsubstituted 2 to 8membered heteroalkyl, halogen, CN, CF₃ or OCF₃; R¹, R², and Y are asdescribed above in the discussion of the modulator compositions.

II. Assays for Modulators of KCNQ Channels

Assays for determining the ability of a compound of the invention toopen a potassium ion channel are generally known in the art. One ofskill in the art is able to determine an appropriate assay forinvestigating the activity of a selected compound of the inventiontowards a particular ion channel. For simplicity, portions of thefollowing discussion focuses on KCNQ2 as a representative example,however, the discussion is equally applicable to other potassium ionchannels.

KCNQ monomers as well as KCNQ alleles and polymorphic variants aresubunits of potassium channels. The activity of a potassium channelcomprising KCNQ subunits can be assessed using a variety of in vitro andin vivo assays, e.g., measuring current, measuring membrane potential,measuring ion flux, e.g., potassium or rubidium, measuring potassiumconcentration, measuring second messengers and transcription levels,using potassium-dependent yeast growth assays, and using e.g.,voltage-sensitive dyes, radioactive tracers, and patch-clampelectrophysiology.

Furthermore, such assays can be used to test for activators of channelscomprising KCNQ. As discussed elsewhere herein, activators (openers) ofa potassium channel are useful for treating various disordersattributable to potassium channels. Such modulators are also useful forinvestigation of the channel diversity provided by KCNQ and theregulation/modulation of potassium channel activity provided by KCNQ.

Putative modulators of the potassium channels are tested usingbiologically active KCNQ, either recombinant or naturally occurring, orby using native cells, like cells from the nervous system expressing theM-current. KCNQ can be isolated, co-expressed or expressed in a cell, orexpressed in a membrane derived from a cell. In such assays, KCNQ2 isexpressed alone to form a homomeric potassium channel or is co-expressedwith a second subunit (e.g., another KCNQ family member, preferablyKCNQ3) so as to form a heteromeric potassium channel. Modulation istested using one of the in vitro or in vivo assays described above.Samples or assays that are treated with a potential potassium channelactivator are compared to control samples without the test compound, toexamine the extent of modulation. Control samples (untreated withactivators) are assigned a relative potassium channel activity value of100. Activation of channels comprising KCNQ2 is achieved when thepotassium channel activity value relative to the control is 110%, morepreferably 130%, more preferably 170% higher. Compounds that increasethe flux of ions will cause a detectable increase in the ion currentdensity by increasing the probability of a channel comprising KCNQ2being open, by decreasing the probability of it being closed, byincreasing conductance through the channel, and/or increasing the numberor expression of channels.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the potassium channel comprising, for example, KCNQ2, KCNQ2/3or the M-current. A preferred means to determine changes in cellularpolarization is by measuring changes in current or voltage with thevoltage-clamp and patch-clamp techniques, using the “cell-attached”mode, the “inside-out” mode, the “outside-out” mode, the “perforatedcell” mode, the “one or two electrode” mode, or the “whole cell” mode(see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)).Whole cell currents are conveniently determined using the standardmethodology (see, e.g., Hamil et al., Pflugers. Archiv. 391:85 (1981).Other known assays include: radiolabeled rubidium flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J.Membrane Biology 137:59-70 (1994)). Assays for compounds capable ofincreasing potassium flux through M-current channels found in nativecells or through the channel proteins comprising KCNQ2 orheteromultimers of KCNQ subunits can be performed by application of thecompounds to a bath solution in contact with and comprising cells havinga channel of interest (see, e.g., Blatz et al., Nature 323:718-720(1986); Park, J. Physiol. 481:555-570 (1994)). Generally, the compoundsto be tested are present in the range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of ions such as potassium or rubidium ions. The cations can bemeasured in a variety of standard ways. They can be measured directly byconcentration changes of the ions or indirectly by membrane potential orby radio-labeling of the ions. Consequences of the test compound on ionflux can be quite varied. Accordingly, any suitable physiological changecan be used to assess the influence of a test compound on the channelsof this invention. The effects of a test compound can be measured by atoxin binding assay. When the functional consequences are determinedusing intact cells or animals, one can also measure a variety of effectssuch as transmitter release (e.g., dopamine), hormone release (e.g.,insulin), transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), cell volume changes (e.g., inred blood cells), immunoresponses (e.g., T cell activation), changes incell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as Ca²⁺, or cyclic nucleotides.

KCNQ2 orthologs will generally confer substantially similar propertieson a channel comprising such KCNQ2, as described above. In a preferredembodiment, the cell placed in contact with a compound that is suspectedto be a KCNQ2 homolog is assayed for increasing or decreasing ion fluxin a eukaryotic cell, e.g., an oocyte of Xenopus (e.g., Xenopus laevis)or a mammalian cell such as a CHO or HeLa cell. Channels that areaffected by compounds in ways similar to KCNQ2 are considered homologsor orthologs of KCNQ2.

Utilizing screening assays such as described above, compounds of theinvention were tested for their ability to open voltage-gated potassiumchannels. The results of these assays are set forth in Table 1 in whichthe data are presented in terms of relative potency of the compoundstested to one another. The compound numbers in Table 1 arecross-referenced to the compounds displayed in FIG. 1

III. Pharmaceutical Compositions of Potassium Channel Modulators

In another aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and acompound of Formulae (I)-(V) provided above.

Formulation of the Compounds (Compositions)

The compounds of the present invention can be prepared and administeredin a wide variety of oral, parenteral and topical dosage forms. Thus,the compounds of the present invention can be administered by injection,that is, intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally. Also, thecompounds described herein can be administered by inhalation, forexample, intranasally. Additionally, the compounds of the presentinvention can be administered transdermally. Accordingly, the presentinvention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier or excipient and either a compoundof Formula I, or a pharmaceutically acceptable salt thereof.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from 5% or 10% to 70% of theactive compound. Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Effective Dosages

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For example, when administered in methods to treat pain oranxiety, such compositions will contain an amount of active ingredienteffective to achieve a clinically relevant degree of reduction in thecondition being treated. Similarly, when the pharmaceutical compositionis used to treat or prevent a central or peripheral nervous systemdisorder, e.g., Parkinson's disease a therapeutically effective amountwill reduce one or more symptoms characteristic of the diseases (e.g.,tremors) to below a predetermined pressure threshold. Determination of atherapeutically effective amount of a compound of the invention is wellwithin the capabilities of those skilled in the art, especially in lightof the detailed disclosure herein.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Target plasmaconcentrations will be those concentrations of active compound(s) thatare capable of modulating, e.g., activating or opening the KCNQ channel.In preferred embodiments, the KCNQ channel activity is altered by atleast 30%. Target plasma concentrations of active compound(s) that arecapable of inducing at least about 50%, 70%, or even 90% or higheralteration of the KCNQ channel potassium flux are presently preferred.The percentage of alteration of the KCNQ channel in the patient can bemonitored to assess the appropriateness of the plasma drug concentrationachieved, and the dosage can be adjusted upwards or downwards to achievethe desired percentage of alteration.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a circulating concentration thathas been found to be effective in animals. A particularly useful animalmodel for predicting anticonvulsant dosages is the maximal electroshockassay (Fischer R S, Brain Res. Rev. 14: 245-278 (1989)). The dosage inhumans can be adjusted by monitoring KCNQ channel activation andadjusting the dosage upwards or downwards, as described above.

A therapeutically effective dose can also be determined from human datafor compounds which are known to exhibit similar pharmacologicalactivities, such as retigabine (Rudnfeldt et al., Neuroscience Lett.282: 73-76 (2000)).

Adjusting the dose to achieve maximal efficacy in humans based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

By way of example, when a compound of the invention is used in theprophylaxis and/or treatment of an exemplary disease such as epilepsy, acirculating concentration of administered compound of about 0.001 μM to20 μM is considered to be effective, with about 0.01 μM to 5 μM beingpreferred.

Patient doses for oral administration of the compounds described herein,which is the preferred mode of administration for prophylaxis and fortreatment of an exemplary disease such as epilepsy, typically range fromabout 1 mg/day to about 10,000 mg/day, more typically from about 10mg/day to about 1,000 mg/day, and most typically from about 1 mg/day toabout 500 mg/day. Stated in terms of patient body weight, typicaldosages range from about 0.01 to about 150 mg/kg/day, more typicallyfrom about 0.1 to about 15 mg/kg/day, and most typically from about 0.5to about 10 mg/kg/day.

For other modes of administration, dosage amount and interval can beadjusted individually to provide plasma levels of the administeredcompound effective for the particular clinical indication being treated.For example, if acute epileptic seizures are the most dominant clinicalmanifestation, in one embodiment, a compound according to the inventioncan be administered in relatively high concentrations multiple times perday. Alternatively, if the patient exhibits only periodic epilepticseizures on an infrequent, periodic or irregular basis, in oneembodiment, it may be more desirable to administer a compound of theinvention at minimal effective concentrations and to use a less frequentadministration regimen. This will provide a therapeutic regimen that iscommensurate with the severity of the individual's disease.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

Therapeutic Index

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

IV. Methods for Increasing Ion Flow in Voltage-Dependent PotassiumChannels

In yet another aspect, the present invention provides methods forincreasing ion flow through voltage dependent potassium channels in acell. The method includes contacting a cell containing the target ionchannels with an amount of a compound of the invention sufficient toenhancer the activity of a potassium channel.

The methods provided in this aspect of the invention are useful for thediagnosis of conditions that can be treated by modulating ion fluxthrough voltage-dependent potassium channels, or for determining if apatient will be responsive to therapeutic agents, which act by openingpotassium channels. In particular, a patient's cell sample can beobtained and contacted with a compound of the invention and the ion fluxcan be measured relative to a cell's ion flux in the absence of acompound of the invention. An increase in ion flux will typicallyindicate that the patient will be responsive to a therapeutic regimen ofion channel openers.

V. Methods for Treating Conditions Mediated by Voltage-DependentPotassium Channels

In still another aspect, the present invention provides a method for thetreatment of a central or peripheral nervous system disorder orcondition through modulation of a voltage-dependent potassium channel.In this method, a subject in need of such treatment is administered aneffective amount of a compound having the formula provided above.

The compounds provided herein are useful as potassium channel modulatorsand find therapeutic utility via modulation of voltage-dependentpotassium channels in the treatment of diseases or conditions. Thepotassium channels targets for the compounds of the invention aredescribed herein as voltage-dependent potassium channels such as theKCNQ potassium channels. As noted above, these channels may includehomomultimers and heteromultimers of KCNQ2, KCNQ3, KCNQ4, KCNQ5 andKCNQ6. A heteromultimer of two proteins, e.g., KCNQ2 and KCNQ3 isreferred to as, for example, KCNQ2/3, KCNQ3/5, etc. The conditions thatcan be treated with the compounds and compositions of the presentinvention may include, but are not limited to, central or peripheralnervous system disorders (e.g., migraine, ataxia, Parkinson's disease,bipolar disorders, trigeminal neuralgia, spasticity, mood disorders,brain tumors, psychotic disorders, myokymia, seizures, epilepsy, hearingand vision loss, Alzheimer's disease, age-related memory loss, learningdeficiencies, anxiety, and motor neuron diseases). The compounds andcompositions of the present invention may also serve as neuroprotectiveagents (e.g., to prevent stroke and the like). In a preferredembodiment, the condition or disorder to be treated is epilepsy orseizures. In another preferred embodiment, the condition or disorder ishearing loss.

In therapeutic use for the treatment of epilepsy or other neurologicalconditions, the compounds utilized in the pharmaceutical method of theinvention are administered at the initial dosage of about 0.001 mg/kg toabout 1000 mg/kg daily. A daily dose range of about 0.1 mg/kg to about100 mg/kg is more typical. The dosages, however, may be varied dependingupon the requirements of the patient, the severity of the conditionbeing treated, and the compound being employed. Determination of theproper dosage for a particular situation is within the skill of thepractitioner. Generally, treatment is initiated with smaller dosages,which are less than the optimum dose of the compound. Thereafter, thedosage is increased by small increments until the optimum effect undercircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day, if desired.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

In the examples below, unless otherwise stated, temperatures are givenin degrees Celsius (° C.); operations were carried out at room orambient temperature (typically a range of from about 18-25° C.;evaporation of solvent was carried out using a rotary evaporator underreduced pressure (typically, 4.5-30 mmHg) with a bath temperature of upto 60° C.; the course of reactions was typically followed by TLC andreaction times are provided for illustration only; melting points areuncorrected; products exhibited satisfactory ¹H-NMR and/ormicroanalytical data; yields are provided for illustration only; and thefollowing conventional abbreviations are also used: mp (melting point),L (liter(s)), mL (milliliters), mmol (millimoles), g (grams), mg(milligrams), min (minutes), and h (hours).

Example 1

Example 1 sets forth a general method for preparing 3-amino-substitutedfused ring heterocycles from anilines according to Scheme 1.

1.1 Preparation of 2-(Trifluoromethoxy)-α-oximinoanilide

According to Scheme 8, hydroxylamine sulfate (18 g, 3 equivalents) wasdissolved in water (80 mL) and then chloral hydrate (7.3 g, 1.2equivalents) was added. After 15 min, a solution of the starting aniline(5.0 ml., 37 mmol), dissolved in 1 N HCl (44 mL, 1.2 equivalents), wasadded dropwise (20 min) to the reaction mixture. The resulting cloudymixture was heated at 80° C. for 1 h, cooled to rt, and diluted withCHCl₃ (160 mL). The aqueous layer was extracted with CHCl₃ (2×80 mL).The combined organic layers were dried (Na₂SO₄), filtered andevaporated. Hexanes (40 mL) were added. Evaporation of the solventprovided crude product which was used in the next step without furtherpurification: MS(ESI): 247 (M−H)⁻.

1.2 Preparation of 7-(Trifluoromethoxy)isatin

According to Scheme 9, concentrated sulfuric acid (40 mL) was heated to65° C. and poured into a flask containing the crude2-(trifluoromethoxy)-α-oximinoanilide from Example 1.1. The mixture wasstirred at 65° C. until homogeneous and then the temperature wasincreased to 80° C. After 90 min, the resulting black mixture was pouredinto ice/water (300 mL) and diluted with 15% i-PrOH/CHCl₃ (200 mL).After slow addition of 6 N NaOH (80 mL, 15 mins), the aqueous layer wasextracted with CHCl₃ (2×80 mL). To the combined organic layers was addedsilica gel (30 g) and the solvent was then evaporated. The resultingsolid was applied to a column of silica gel and eluted with 15-25%EtOAc/hexanes to provide the product (5.0 g, 59%, 2 steps): MS(ESI): 230(M−H)⁻.

1.3 Preparation of 2-Amino-3-trifluoromethoxybenzoic Acid HydrogenChloride

According to Scheme 10, the starting isatin (3.5 g, 15 mmol) and 6 NNaOH (25 mL, 10 equivalents) were heated at 80° C. and treated slowlywith 30% H₂O₂ (4.5 mL, 0.3 mL/mmol, gas evolution). After 1 h, themixture was cooled to room temperature and 6 N HCl was added dropwise(27 mL), whereupon a precipitate formed. After 30 mins, the mixture wascooled to 0° C. and the solid was filtered, washed with ice cold water(15 mL) and dissolved in CH₂Cl₂ (45 mL). The organic solution was dried(Na₂SO₄), filtered and evaporated to provide the product as an off-whitesolid (3.4 g, 88%): MS(ESI): 220 (M−H)⁻.

Preparation of 3-Amino-2-ethyl-8-trifluoromethoxy-3H-quinazolin-4-one

According to Scheme 11, the starting anthranilic acid (2.1 g, 8.1 mmol)was dissolved in dry THF (40 mL) and treated with pyridine (3.9 mL, 6equivalents), followed by propionyl chloride (2.9 mL, 4 equivalents,immediate precipitate). The mixture was heated at reflux for 15 h andthen cooled to 0° C. before adding hydrazine (3.1 mL, 12 equivalents).This mixture was then heated at reflux for 3 h. Most of the THF wasevaporated and the resulting mixture diluted with CHCl₃ (40 mL),saturated aqueous NaHCO₃ solution (60 mL) and water (20 mL). The aqueouslayer was extracted with CHCl₃ (2×20 mL). Silica gel (15 g) was added tothe combined organic layers and the solvent was then evaporated. Theresulting solid was applied to a column of silica gel and eluted with5-8% MeCN/45-42% hexanes/50% CH₂Cl₂ to provide the product as anoff-white solid (2.2 g, 99%): MS(ESI): 274 (MH⁺).

Example 2 2.1 General Preparative Method for Urea Fused RingHeterocycles

As shown in Scheme 12, the starting 3-aminoquinazolinone (0.1 mmol) wasdissolved in dry CH₂ClCH₂Cl (1 mL) and treated with pyridine (1.5equivalents) followed by an isocyanate (1.2 equivalents). After 15 h,saturated NaHCO₃ solution (0.6 mL) was added and 30 min later, themixture was diluted with CHCl₃ (1 mL) and water (0.4 mL). The organiclayer was washed with water (0.4 mL) and the solvent was removed using aspeedvac. The urea product was typically obtained as a solid in goodyield (>90%) and with high purity (>95% by LC/MS). In some cases, theproduct was purified by flash chromatography, using EtOAc/hexanes as theeluent.

2.2 Results1-(2-Cyclohexyl-4-oxo-4H-quinazolin-3-yl)-3-(2-fluorobenzyl)-urea

¹H NMR (CDCl₃) δ 8.10 (d, J=8.0 Hz, 1H), 7.6-7.7 (m, 2H), 6.9-7.4 (m,5H), 4.39 (d, J=33.7 Hz, 2H), 2.9-3.1 (m, 1H), 2.50 (t, J=7.5 Hz, 2H),1.6-2.0 (m, 6H), 1.2-1.6 (m, 4H); MS(ESI): 395 (M+H)⁺.

Example 3 3.1a General Preparative Method for Amide Quinazolines

According to Scheme 13, the starting 3-aminoquinazolinone (0.1 mmol) wasdissolved in dry THF (1 mL) and treated with pyridine (1.5 equivalents)followed by an acid chloride (1.2 equivalents). After 15 h, saturatedNaHCO₃ solution (0.6 mL) was added and 30 min later, the mixture wasdiluted with EtOAc (2 mL) and water (0.4 mL). The organic layer waswashed with water (0.4 mL). Removal of the solvent with a speedvactypically provided the amide product as a solid in good yield (>90%) andwith high purity (>95% by LC/MS). In some cases, the product waspurified by flash chromatography, using EtOAc/hexanes as the eluent.

3.1b Alternate General Preparative Method for Amide Fused RingHeterocycles

Scheme 14 sets forth a method in which a carboxylic acid (1.2equivalents) was dissolved in dry THF (1 mL) and treated with dry DMF (1drop) followed by oxalyl chloride (1.3 equivalents; gas evolution; mildexotherm). After 1 h, pyridine (1 equivalent) was added (immediateprecipitate), followed by the 3-aminoquinazolinone (0.1 mmol) and thenmore pyridine (2 equivalents). After 15 h, saturated NaHCO₃ solution(0.6 mL) was added and 30 min later, the mixture was diluted with EtOAc(2 mL) and water (0.4 mL). The organic layer was washed with water (0.4mL). Removal of the solvent with a speedvac typically provided the amideproduct as a solid in good yield (>90%) and with high purity (>95% byLC/MS). In some cases, the product was purified by flash chromatography,using EtOAc/hexanes as the eluent.

3.1c General Procedure for Preparing Isatoic Anhydrides

As set forth in Scheme 6, anhydrous pyridine (2 eq) was added to asolution of anthranilic acid (1 eq) in dry methylene chloride andacetonitrile (1:1, 40 mL/g of 2-aminobenzoic acid) at room temperature.Solid triphosgene (⅓ eq) was then added in one portion and the resultingmixture was heated at 50° C. for 2 h. The resulting solid was collectedby filtration and dried in vacuo. The crude isatoic anhydrides (VIII)were typically obtained in 50-80% yields. Though contaminated with somepyridinium hydrochloride, the anhydrides were used in the next stepwithout further purification.

3.1d General Procedure for Preparing Acylhydrazides

A mixture of isatoic anhydride (1 eq) and appropriate phenylacetylhydrazide (1.1 eq) were heated in glacial AcOH (4 ml/mmol) at 50° C. for2-6 h. The resulting solution was cooled and water was added whileshaking. The white precipitate was collected by filtration, washed withwater and dried in vacuo at 50° C. for 4 h. The desired products wereobtained as white solids in high purity (typically >90%) and moderateyields (typically 45-60%).

3.1e General Procedure for Preparing Nitro-benzoic-N′-phenylacetylHydrazides

The appropriate nitro-benzoic acid derivative (1 eq) was stirred in drymethylene chloride (100 mL/g of acid) at room temperature and to thiswas added two drops of N,N-dimethylformamide (DMF). Neat oxalyl chloride(2 eq) was then added to the mixture drop-wise at such a rate as tocontrol gas evolution. After stirring for 2 h, the volatiles are removedby rotary evaporation and the remaining material was re-dissolved in drymethylene chloride (100 mL/g of acid). Pyridine (2 eq) and theappropriate hydrazide derivative (1 eq) were added consecutively and themixture was allowed to stir at room temperature until the reaction isjudged to be complete by HPLC analysis whereupon the reaction mixturewas poured into water. The organic layer was removed and the water layerwas extracted three times with ethyl acetate. The combined organiclayers were dried (Na₂SO₄) and concentrated to provide the desirednitro-benzoic-N′-phenylacetyl hydrazides in high purity (typically >95%)and yields ranging from 40 to 90%.

3.1f General Procedure for Preparing 2-Amino-benzoic AcidN′-phenylacetyl-hydrazides from Nitro-benzoic-N′-phenylacetyl Hydrazides

Hydrogen (1 Atm) was applied to a mixture ofnitro-benzoic-N′-phenylacetyl hydrazide in methanol (100 mL/g ofhydrazide) and 10% palladium on activated carbon (100 mg/g ofhydrazide). The reaction mixture was stirred at rt for 1-10 h. Theresulting mixture was filtered through Celite/silica gel andconcentrated under reduced pressure. The desired 2-amino-benzoic acidN′-phenylacetyl hydrazides were, in general, used directly in the nextstep without any further purification. In those instances when thedesired products were contaminated, the hydrazides were purified bysilica gel chromatography using EtOAc/hexanes.

3.2 Results

N-(2-Cyclohexyl-4-oxo-4H-quinazolin-3-yl)-3,3-dimethyl-butyramide

¹H NMR (CDCl₃) δ 8.19 (d, J=8.0 Hz, 1H), 7.6-7.8 (m, 2H), 7.4-7.5 (m,1H), 2.8-2.9 (m, 1H), 2.36 (s, 3H), 1.2-2.0 (m, 10H), 1.14 (s, 9H);MS(ESI): 342 (M+H)⁺.

N-(2-Cyclohexyl-4-oxo-4H-quinazolin-3-yl)-3-cyclopentyl-propionamide

¹H NMR (CDCl₃) δ 8.18 (dd, J=1.0, 8.0 Hz, 1H), 7.6-7.8 (m, 2H), 7.3-7.5(m, 1H), 2.7-2.9 (m, 1H), 2.50 (t, J=7.5 Hz, 2H), 1.2-2.0 (m, 21H);MS(ESI): 368 (M+H)⁺.

N-(2-Isopropyl-4-oxo-4H-quinazolin-3-yl)-2-(3-trifluoromethoxyphenyl)-acetamide

¹H NMR (CDCl₃) δ 8.18 (d, J=8.0 Hz, 1H), 7.6-7.8 (m, 2H), 7.1-7.5 (m,5H), 3.8-4.0 (m, 2H), 3.00 (h, J=6.8 Hz, 1H), 1.25 (d, J=6.7 Hz, 3H),1.18 (d, J=6.8 Hz, 3H); MS(ESI): 406 (M+H)⁺.

1-(2-Cyclohexyl-4-oxo-4H-quinazolin-3-yl)-3-(2-fluorobenzyl)-urea

¹H NMR (CDCl₃) δ 8.10 (d, J=8.0 Hz, 1H), 7.6-7.7 (m, 2H), 6.9-7.4 (m,5H), 4.39 (d, J=33.7 Hz, 2H), 2.9-3.1 (m, 1H), 2.50 (t, J=7.5 Hz, 2H),1.6-2.0 (m, 6H), 1.2-1.6 (m, 4H); MS(ESI): 395 (M+H)⁺.

N-(2-Ethyl-7-fluoro-4-oxo-4H-quinazolin-3-yl)-2-(4-fluorophenyl)-acetamide

¹H NMR (CDCl₃) δ 8.19 (dd, J=6.1, 8.7 Hz, 1H), 7.0-7.4 (m, 6H), 3.7-3.9(m, 2H), 2.67 (q, J=7.3 Hz, 2H), 1.25 (t, J=7.3 Hz, 3H); MS(ESI): 344(M+H)⁺.

N-(7-Fluoro-4-oxo-(2-tetrahydrofuran-3-yl)-4H-quinazolin-3-yl)-2-(4-fluorophenyl)-acetamide

¹H NMR (CDCl₃) δ 8.1-8.2 (m, 1H), 7.0-7.5 (m, 6H), 3.7-4.1 (m, 6H),3.4-3.6 (m, 1H), 2.0-2.3 (m, 2H); MS(ESI): 386 (M+H)⁺.

N-(2-Cyclopropyl-4-oxo-7-trifluoromethyl-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.23 (d, J=8.4 Hz, 1H), 7.82 (s, 1H), 7.54 (d, J=8.5Hz, 1H), 7.3-7.5 (m, 5H), 3.8-4.0 (m, 2H), 2.0-2.1 (m, 1H), 1.2-1.3 (m,2H), 0.9-1.0 (m, 2H); MS(ESI): 388 (M+H)⁺.

N-(2-Ethyl-4-oxo-7-trifluoromethoxy-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.21 (d, J=8.8 Hz, 1H), 7.50 (s, 1H), 7.2-7.4 (m, 6H),3.8-4.0 (m, 2H), 2.68 (q, J=7.3 Hz, 2H), 1.25 (t, J=7.4 Hz, 3H);MS(ESI): 392 (M+H)⁺.

N-(2-Methyl-4-oxo-8-trifluoromethyl-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.36 (d, J=7.9 Hz, 1H), 8.05 (d, J=7.7 Hz, 1H), 7.3-7.5(m, 6H), 3.8-4.0 (m, 2H), 2.47 (s, 3H); MS(ESI): 362 (M+H)⁺.

N-(2-Ethyl-4-oxo-8-trifluoromethyl-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.36 (d, J=8.0 Hz, 1H), 8.05 (d, J=7.5 Hz, 1H), 7.3-7.5(m, 6H), 3.8-4.0 (m, 2H), 2.69 (q, J=7.3 Hz, 2H), 1.28 (t, J=7.3 Hz,3H); MS(ESI): 376 (M+H)⁺.

N-(2-Cycloproyl-4-oxo-8-trifluoromethyl-4H-quinazolin-3-yl)-2-(3,4-difluorophenyl)-acetamide

¹H NMR (CDCl₃) δ 8.33 (d, J=7.3 Hz, 1H), 8.01 (d, J=7.4 Hz, 1H), 7.43(t, J=7.8, Hz, 1H), 7.1-7.3 (m, 3H), 3.83 (s, 2H), 2.0-2.1 (m, 1H),1.3-1.4 (m, 2H), 1.0-1.1 (m, 2H); MS(ESI): 424 (M+H)⁺.

N-(2-Cycloproyl-4-oxo-8-trifluoromethyl-4H-quinazolin-3-yl)-3-(3-fluorophenyl)-propionamide

¹H NMR (CDCl₃) δ 8.32 (d, J=8.0 Hz, 1H), 8.00 (d, J=7.5 Hz, 1H), 7.41(t, J=7.6, Hz, 1H), 7.2-7.3 (m, 1H), 6.8-7.1 (m, 3H), 3.8-3.2 (m, 2H),2.7-2.9 (m, 2H), 1.8-1.9 (m, 1H), 1.2-1.4 (m, 2H), 0.9-1.1 (m, 2H);MS(ESI): 420 (M+H)⁺.

N-(2-Ethyl-4-oxo-8-trifluoromethoxy-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.13 (dd, J=1.4, 8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H),7.3-7.5 (m, 6H), 3.8-4.0 (m, 2H), 2.70 (q, J=7.3 Hz, 2H), 1.27 (t, J=7.3Hz, 3H); MS(ESI): 392 (M+H)⁺.

2-(3-Fluorophenyl)-N-(4-oxo-2-propyl-8-trifluoromethoxy-4H-quinazolin-3-yl)-acetamide

¹H NMR (CDCl₃) δ 8.14 (dd, J=1.4, 8.1 Hz, 1H), 7.65 (d, J=8.0 Hz, 1H),7.3-7.5 (m, 2H), 7.0-7.2 (m, 3H), 3.8-4.0 (m, 2H), 2.63 (t, J=7.5 Hz,2H), 1.7-1.9 (m, 2H), 0.96 (t, J=7.5 Hz, 3H); MS(ESI): 424 (M+H)⁺.

N-(2-(1,1-Difluoroethyl)-4-oxo-8-trifluoromethyl-4H-quinazolin-3-yl)-2-phenyl-acetamide:

¹H NMR (CDCl₃) δ 8.40 (d, J=7.8 Hz, 1H), 8.07 (d, J=7.7 Hz, 1H), 7.60(dd, J=7.7, 7.7 Hz, 1H), 7.2-7.4 (m, 5H), 3.76 (s, 2H), 2.03 (t, J=19.1Hz, 3H); MS(ESI): 412 (M+H)⁺.

N-(8-Chloro-2-(1,1-difluoroethyl)-4-oxo-4H-quinazolin-3-yl)-2-(4-fluorophenyl)-acetamide:

¹H NMR (CDCl₃) δ 8.11 (dd, J=2.5, 8.0 Hz, 1H), 7.84 (dd, J=2.2, 7.6 Hz,1H), 7.44 (ddd, J=3.7, 8.0, 8.0 Hz, 1H), 7.2-7.4 (m, 2H), 6.99 (m, 2H),3.71 (d, J=3.3 Hz, 2H), 2.06 (dt, J=3.5, 19.1 Hz, 3H); MS(ESI): 396(M+H)⁺.

Example 4 4.1 General Procedure for Preparing Substituted Fused RingHeterocycles

Acid chloride (1 eq) was added to a solution of 2-amino-benzoic acidN′-phenylacetyl hydrazide (1 eq) and pyridine (1 eq) in anhydrousdioxane (5 mL/1 mmol) at rt. The mixture was heated at 50° C. for 1 h(or until LCMS indicated that the acylation complete). 4N HCl in dioxane(0.1 ml/mmol) was added and the solution heated at 60-70° C. for 2-6 h.The solvent was removed under reduced pressure and the desiredsubstituted fused ring heterocycles were purified by columnchromatography (EtOAc/hexanes). The compounds were obtained as off-whitesolids in high purity (>95% LC) and moderate to high yields (55-95%).

4.2 Results

N-(2-tert-Butyl-7-fluoro-4-oxo-4H-quinazolin-3-yl)-2-(4-fluorophenyl)-acetamide

¹H NMR (CDCl₃) δ 8.14 (dd, J=6.3, 8.9 Hz, 1H), 7.70 (brs, 1H), 7.40 (dd,J=5.2, 8.3 Hz, 2H), 7.32 (dd, J=2.1, 9.8 Hz, 1H), 7.18-7.07 (m, 3H),3.91-3.77 (m, 2H), 1.32 (s, 9H); MS(ESI): 372 (M+H)⁺.

N-(2-Ethyl-8-methyl-4-oxo-4H-quinazolin-3-yl)-2-(4-fluorophenyl)-acetamide

¹H NMR (CDCl₃) δ 8.66 (brs, 1H), 7.99 (d, J=7.8 Hz, 1H), 7.58 (d, J=7.8Hz, 1H), 7.38-7.30 (m, 3H), 7.03 (t, J=8.6 Hz, 2H), 3.86-3.72 (m, 2H),2.72-2.60 (m, 2H), 2.58 (s, 3H), 1.26 (t, J=7.3, 3H); MS(ESI): 340(M+H)⁺.

N-(8-Chloro-2-ethyl-4-oxo-4H-quinazolin-3-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.70 (brs, 1H), 8.03 (dd, J=1.2, 8.0 Hz, 1H), 7.81 (dd,J=1.2, 8.0 Hz, 1H), 7.38-7.30 (m, 3H), 7.12-6.98 (m, 3H), 3.83-3.70-(m,2H), 2.78-2.67 (q, J=7.3 Hz, 2H), 1.26 (t, J=7.3, 3H); MS(ESI): 361(M+H)⁺.

Example 5 5.1 General Procedure for Preparing1,1-Dioxo-1H-1λ⁶-benzo[1,2,4]thiadiazin-2-yl Derivatives

Substituted 2-nitro-sulfonyl chloride was added drop-wise to a stirringsolution of hydrazide and pyridine in THF at rt. After 1 h the solventwas removed and the residue was purified by passage through a shortsilica plug (EtOAc/hexane; 1:2). The concentrated product was dissolvedin anhydrous methanol (5 ml/mmol) and anhydrous Na₂SO₄ was added (˜200mg/mmol). 10% Pd/C was added (10% w/w) and 1 atm of hydrogen wasapplied. The mixture was stirred for 4 h after which the mixture wasfiltered through a mixture of silica/celite (1:1 EtOAc/hexanes).Concentration of the eluent gave the crude anilines as solids in highpurity (>95% LCMS) and high yield (80-90%). Treatment of the anilineswith the appropriate orthoester in an anhydrous acidic medium atelevated temperatures (60-100° C.) gave the desired cyclized products.Purification by column chromatography afforded the products as off-whitesolids in high yield (>80%) and purity (>95%).

5.2 Results

N-(3-Ethyl-1,1-dioxo-1H-1λ⁶-benzo[1,2,4]thiadiazin-2-yl)-2-phenyl-acetamide

¹H NMR (CDCl₃) δ 8.31 (brs, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.68-7.66 (m,2H), 7.48-7.35 (m, 6H), 3.78 (s, 2H), 2.55 (q, J=7.3 Hz, 2H), 1.18 (t,J=7.3 Hz, 3H); MS(ESI): 344 (M+H)⁺.

Example 6 6.1 General Procedure for Preparing 5-Membered Fused RingHeterocycles

Ethyl ester p (0.003 mol) was added to a solution of KOH (1.1 eq, 0.0033mol) in water (4 ml) at room temperature. The resulting mixture washeated at 100° C. (sealed vial) while stirring. After 15 min thereaction was checked by LCMS. If starting material remained a further0.1 eq of KOH was added and heating continued for 20 min. Reaction wascooled to 0° C. whereupon 6N HCl(aq) was added until pH˜3 was attained.The resulting white solid q was collected by filtration and dried invacuo (50° C.) for 3 h. Yields were typically 40-70% with puritiestypically >95%.

BOP (1 eq) was added to a suspension of acid q (1 eq), hydrazide (1.5eq) and Et3N (3 eq) in EtOAc/THF (2:1, 6 ml per mmol). The resultingsuspension was shaken at room temperature overnight. Solvent was removedand EtOAc (6 ml/mmol) was added. The mixture was warmed until a clearsolution formed. Washed with water (2×3 ml), saturated NaHCO3 (aq) (2×3ml), saturated NH4Cl (2×3 ml) then water again (3 ml). Hexane (6 ml) wasadded to the organic layer. Precipitate was collected by filtration anddried in vacuo. If no precipitate formed the reaction was concentratedand purified by column chromatography (Hexanes/EtOAc). Products wereafforded as white solid in yields 60-85% with purities >95%.

Alternative methods were used to form the 5-membered fused ringheterocycle s using either orthoester or acid chloride. In the former,the corresponding orthoester (0.2 ml) and a catalytic amount of pTSA (˜5mg) was added to a solution of r (0.1 mmol) in dioxane (0.5 ml) wasadded. The solution was heated at 80° C. until conversion was complete(typically 3-8 h). Solvent was removed under reduced pressure and theproducts were purified by column chromatography (Hexanes/EtOAc).Products were afforded as white solids in yields 60-90% with purities>95%.

Alternativley, the desired carbonyl chloride (1.3 eq) and pyridine (1eq) was added to a solution of r (0.1 mmol) in dioxane (0.5 ml). Thereaction was stirred and heated at 55° C. for 3 h. 0.5 ml of 1N HCl inether (or 0.1 ml of 4N HCl in dioxane) was added and the reaction heatedat 100° C. until complete. Solvent was removed under reduced pressureand the products were purified by column chromatography (Hexanes/EtOAc).Products were afforded as white solids in yields 20-70% with purities>95%.

6.2 Results

5-Amino-1-(2,2,2-trifluoro-ethyl)-1H-pyrazole-4-carboxylic acidN′-phenylacetyl-hydrazide:

¹H NMR (d₆-DMSO) δ 9.92 (brs, 1H), 9.62 (brs, 1H), 7.76 (s, 1H),7.31-7.21 (m, 5H), 6.59 (brs, 2H), 4.88 (q, J=9.1 Hz, 2H), 3.47 (s, 2H);¹⁹F NMR (d₆-DMSO) δ−69.2 (t, J=8.6 Hz, 3F); MS(ESI): 342 (M+H)⁺.

5-Amino-1-(2,2,2-trifluoro-ethyl)-1H-pyrazole-4-carboxylic acidN′-[2-(4-fluoro-phenyl)-acetyl]-hydrazide:

¹H NMR (d₆-DMSO) δ 9.91 (brs, 1H), 9.62 (brs, 1H), 7.76 (s, 1H),7.36-7.31 (dd, J=8.4 & 5.9 Hz, 2H), 7.15-7.09 (t, J=8.8 Hz, 2H), 6.58(brs, 2H), 4.87 (q, J=9.1 Hz, 2H), 3.47 (s, 2H); MS(ESI): 360 (M+H)⁺.

N-[6-Ethyl-4-oxo-1-(2,2,2-trifluoro-ethyl)-1,4-dihydro-pyrazolo[3,4-d]pyrimidin-5-yl]-2-phenyl-acetamide:

¹H NMR (d₆-DMSO) δ 11.2 (s, 1H), 8.25 (s, 1H), 7.35-7.23 (m, 5H), 5.22(q, J=8.8 Hz, 2H), 3.74 (m, 2H), 3.07-2.44 (m, 2H); ¹⁹F NMR (d₆-DMSO)δ−69.2 (t, J=8.6 Hz, 3F); MS(ESI): 380 (M+H)⁺.

N-[6-Methyl-4-oxo-1-(2,2,2-trifluoro-ethyl)-1,4-dihydro-pyrazolo[3,4-d]pyrimidin-5-yl]-2-phenyl-acetamide:

¹H NMR (d₆-DMSO) δ 11.2 (s, 1H), 8.24 (s, 1H), 7.37-7.25 (m, 5H), 5.21(q, J=9.0 Hz, 2H), 3.72 (m, 2H), 2.31 (s, 3H); ¹⁹F NMR (d₆-DMSO) δ−69.2(t, J=8.5 Hz, 3F); MS(ESI): 366 (M+H)⁺.

Example 7

This example illustrates a screening protocol for evaluating putativepotassium channel agonists for the ability to open voltage-gatedpotassium channels.

7.1 Materials and Methods

NG108-15 cells, a mouse neuroblastoma, rat glioma hybrid cell line,functionally express M-currents (Robbins et al., J. Physiol. 451: 159-85(1992). NG108-15 M-currents are likely comprised, at least in part, ofKCNQ2, KCNQ3 and KCNQ5, since these genes are reportedly robustlyexpressed in differentiated NG108-15 cells (Selyanko et al., J.Neurosci. 19(18): 7742-56 (1999); Schroeder et al., J. Biol. Chem.275(31): 24089-95 (2000)) and KCNQ3 dominant-negative constructs reduceM-current density in these cells (Selyanko et al., J. Neurosci. 22(5):RC212 (2002).

NG108-15 were maintained in DMEM (high glucose) supplemented with 10%fetal bovine serum, 0.05 mM pyridoxine, 0.1 mM hypoxanthine, 400 nMaminopterin, 16 mM thymidine, 50 μgml⁻¹ gentamycin and 10 mM HEPES, inan incubator at 37° C. with a humidified atmosphere of 5% CO₂. Cellswere plated in 96 well plates differentiated by addition of 10 μM PGE1and 50 μM isomethylbutylxanthine to the growth media prior to study.

Differentiated NG108-15 cells were loaded with voltage-sensitive dye byincubation in Earls Balanced Salt Solution (EBSS) containing 5 mM DiBACfor 1 h. Following loading, drug solution containing 5 mM DiBAC wasadded to each well. Changes in fluorescence were measured every 30 s for25 mM. The maximum change in fluorescence was measured and expressed asa percentage of the maximum response obtained in the presence of apositive control agent.

7.2 Results

Table 1 sets forth potencies of representative compounds of theinvention in the NG-108-15 FLIPR assay, for a selection of compounds.

TABLE 1 Compound ID # Activity 1 +++ 10 + 18 +++ 20 +++ 22 +++ 29 ++ 33+++ 35 + 43 ++ 50 +++ 51 +++ 57 +++ 62 ++ 63 +++ 65 +++ 66 + 69 +++ 72 +89 +++ 91 +++ 95 +++ 100 + 110 +++ 134 +++ 159 ++ 166 ++ 169 +++ 180 +++196 ++ 199 + 205 ++ 206 +++ 215 +++ 217 ++ 233 ++ 242 +++ 244 +++ 247 ++271 +++ 273 +++ 278 +++ 293 + 301 +++ 310 ++ 314 +++ 320 +++ 331 ++ 348+++ 354 +++ 358 +++ 359 +++ 360 +++ 364 +++ 365 +++ 366 +++ 384 ++ 385+++ 391 ++ 392 ++ 393 + 416 + 421 + 420 ++ + Represents 10 μM < EC₅₀ < 3μM; ++ represents 3 μM < EC₅₀ < 0.5 μM; +++ represents EC₅₀ < 0.5 μM.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising an effective amount of the compound according toclaim 1 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable excipient.